MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid

Abstract

Since our initial characterization of the iMACs, different di-bromocarbazole derivatives with anti-apoptotic function have been developed and tested in several mouse models of brain injury and neurodegeneration [13-21]. Owing to the increased therapeutic potential of anti-apoptotic di-bromocarbazole derivatives, we sought to expand our knowledge of the mechanism of action of these small molecule inhibitors. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and quantitative western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax oligomers in the outer membrane. A comparison of the inhibitory effects over channel conductance and cytochrome c release suggests that the iMACs interacted with both Bax and Bak with similar kinetics. Interestingly, wild type mitochondria were more susceptible to inhibition than the Bak or Bax knockouts. A quantitative western blot analysis showed that wild type mitochondria had lower steady state levels of Bak, which suggests an uneven stoichiometry of the MAC components.